Molecular insights of nickel binding to therapeutic antibodies as a possible new antibody superantigen

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes

Variable-heavy (VH) families influencing IgA1&2 engagement to the antigen, FcαRI and superantigen proteins G, A, and L

Interest in IgA as an alternative antibody format has increased over the years with much remaining to be investigated in relation to interactions with immune cells. Considering the recent whole antibody investigations showing significant distal effects between the variable (V) and constant (C)- regions that can be mitigated by the hinge regions of both human IgA subtypes A1 and A2, we performed an in-depth mechanistic investigation using a panel of 28 IgA1s and A2s of both Trastuzumab and Pertuzumab models. FcαRI binding were found to be mitigated by the differing glycosylation patterns in IgA1 and 2 with contributions from the CDRs. On their interactions with antigen-Her2 and superantigens PpL, SpG and SpA, PpL was found to sterically hinder Her2 antigen binding with unexpected findings of IgAs binding SpG at the CH2-3 region alongside SpA interacting with IgAs at the CH1. Although the VH3 framework (FWR) is commonly used in CDR grafting, we found the VH1 framework (FWR) to be a possible alternative when grafting IgA1 and 2 owing to its stronger binding to antigen Her2 and weaker interactions to superantigen Protein L and A. These findings lay the foundation to understanding the interactions between IgAs and microbial superantigens, and also guide the engineering of IgAs for future antibody applications and targeting of superantigen-producing microbes

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Therapeutic antibodies have shifted the paradigm of disease treatments from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. Recent findings showed that the antibody constant region can govern localization effects that are useful in reducing side effects due to systemic circulation by the commonly used IgG isotypes. Given their localized mucosal effects, IgA antibodies are increasingly promising therapeutic biologics. While the antibody Fc effector cell activity has been a focus point, recent research showed that the Fc could also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To investigate this, we analysed the IgA antibody constant region and its distal effects on the antigen binding regions using recombinant Pertuzumab IgA1 and IgA2 variants. We found that mutations in the C-region reduced Her2 binding experimentally, and computational structural analysis showed that allosteric communications were highly dependent on the antibody hinge, providing strong evidence that we should consider antibodies as whole proteins rather than a sum of functional regions

Additional file 1: of Structural analyses of 2015-updated drug-resistant mutations in HIV-1 protease: an implication of protease inhibitor cross-resistance

Dynamic changes of pocket volumes of three wild type protease-PI complexes during 10 ns molecular dynamics simulation. The three wild type protease-PI complexes (DRV_0, LPV_0, and NFV_0) contain single residue substitutions S37N, L63P, and V3I respectively that contributed to shrink the PI-binding pocket as compared to the native protease [PDB:1ODW]. The fluctuation of pocket volume of the native protease is estimated based on the three PIs (DRV, LPV, and NFV), and is shown in gray shade (~1955 ± 213 Å3). The molecular dynamics simulations were performed in standard protocol for 2x5ns using AMBER14. (PDF 564 kb

Effect of VH–VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development

Data_Sheet_1.DOCX

<p>Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.</p

Discovery of a novel splice variant of Fcar (CD89) unravels sequence segments necessary for efficient secretion: A story of bad signal peptides and good ones that nevertheless do not make it

<p>The IgA receptor, Fcar (CD89) consists of 5 sequence segments: 2 segments (S1, S2) forming the potential signal peptide, 2 extracellular EC domains that include the IgA binding site, and the transmembrane and cytoplasmic tail (TM/C) region. Numerous Fcar splice variants have been reported with various combinations of the sequence segments mentioned above. Here, we report a novel splice variant termed variant APD isolated from a healthy volunteer that lacks only the IgA-binding EC1 domain. Despite possessing the complete signal peptide S1+S2, the variant APD is only found in the intracellular space whereas the wild-type variant 1 is efficiently secreted and variant 4 leaks to the extracellular space. Further mutational experiments involving signal peptide replacements, cleavage site modifications, and studies on alternative isoforms demonstrate that despite the completeness of the signal peptide motif, the presence of the EC1 domain is essential for efficient extracellular export.</p